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1.  Combining histone deacetylase inhibitors with MDA-7/IL-24 enhances killing of renal carcinoma cells 
Cancer Biology & Therapy  2013;14(11):1039-1049.
In the present study we show that histone deacetylase inhibitors (HDACIs) enhance the anti-tumor effects of melanoma differentiation associated gene-7/interleukin 24 (mda-7/IL-24) in human renal carcinoma cells. Similar data were obtained in other GU tumor cells. Combination of these two agents resulted in increased autophagy that was dependent on expression of ceramide synthase 6, with HDACIs enhancing MDA-7/IL-24 toxicity by increasing generation of ROS and Ca2+. Knock down of CD95 protected cells from HDACI and MDA-7/IL-24 lethality. Sorafenib treatment further enhanced (HDACI + MDA-7/IL-24) lethality. Anoikis resistant renal carcinoma cells were more sensitive to MDA-7/IL-24 that correlated with elevated SRC activity and tyrosine phosphorylation of CD95. We employed a recently constructed serotype 5/3 adenovirus, which is more effective than a serotype 5 virus in delivering mda-7/IL-24 to renal carcinoma cells and which conditionally replicates (CR) in tumor cells expressing MDA-7/IL-24 by virtue of placing the adenoviral E1A gene under the control of the cancer-specific promoter progression elevated gene-3 (Ad.5/3-PEG-E1A-mda-7; CRAd.5/3-mda-7, Ad.5/3-CTV), to define efficacy in renal carcinoma cells. Ad.5/3-CTV decreased the growth of renal carcinoma tumors to a significantly greater extent than did a non-replicative virus Ad.5/3-mda-7. In contralateral uninfected renal carcinoma tumors Ad.5/3-CTV also decreased the growth of tumors to a greater extent than did Ad.5/3-mda-7. In summation, our data demonstrates that HDACIs enhance MDA-7/IL-24-mediated toxicity and tumor specific adenoviral delivery and viral replication of mda-7/IL-24 is an effective pre-clinical renal carcinoma therapeutic.
doi:10.4161/cbt.26110
PMCID: PMC3925659  PMID: 24025359
MDA-7/IL-24; HDACI; ceramide; apoptosis; bystander; cytokine; ROS; caspase; animal study
2.  Sorafenib and pemetrexed toxicity in cancer cells is mediated via SRC-ERK signaling 
Cancer Biology & Therapy  2012;13(9):793-803.
The present studies sought to further understand how the anti-folate pemetrexed and the multi-kinase inhibitor sorafenib interact to kill tumor cells. Sorafenib activated SRC, and via SRC the drug combination activated ERK1/2. Expression of dominant negative SRC or dominant negative MEK1 abolished drug-induced ERK1/2 activation, together with drug-induced autophagy, acidic lysosome formation, and tumor cell killing. Protein phosphatase 2A is an important regulator of the ERK1/2 pathway. Fulvestrant resistant MCF7 cells expressed higher levels of the PP2A inhibitor SET/I2PP2A, had lower endogenous PP2A activity, and had elevated basal ERK1/2 activity compared with their estrogen dependent counterparts. Overexpression of I2PP2A blocked drug-induced activation of ERK1/2 and tumor cell killing. PP2A can be directly activated by ceramide and SET/I2PP2A can be inhibited by ceramide. Inhibition of the de novo ceramide synthase pathway blocked drug-induced ceramide generation, PP2A activation and tumor cell killing. Collectively these findings demonstrate that ERK1/2 plays an essential role downstream of SRC in pemetrexed and sorafenib lethality and that PP2A plays an important role in regulating this process.
doi:10.4161/cbt.20562
PMCID: PMC3679099  PMID: 22673740
ERK; I2PP2A; PP2A; SRC; autophagy; ceramide; pemetrexed; sorafenib
3.  Sorafenib enhances pemetrexed cytotoxicity through an autophagy -dependent mechanism in cancer cells 
Cancer research  2011;71(14):4955-4967.
Pemetrexed (ALIMTA) is a folate anti-metabolite that has been approved for the treatment of non-small cell lung cancer, and has been shown to stimulate autophagy. In the present study, we sought to further understand the role of autophagy in the response to pemetrexed and to test if combination therapy could enhance the level of toxicity through altered autophagy in tumor cells. The multi-kinase inhibitor sorafenib (NEXAVAR), used in the treatment of renal and hepatocellular carcinoma, suppresses tumor angiogenesis and promotes autophagy in tumor cells. We found that sorafenib interacted in a greater than additive fashion with pemetrexed to increase autophagy and to kill a diverse array of tumor cell types. Tumor cell types that displayed high levels of cell killing after combination treatment showed elevated levels of AKT, p70 S6K and/or phosphorylated mTOR, in addition to Class III RTKs such as PDGFRβ and VEGFR1, known in vivo targets of sorafenib. In xenograft and in syngeneic animal models of mammary carcinoma and glioblastoma, the combination of sorafenib and pemetrexed suppressed tumor growth without deleterious effects on normal tissues or animal body mass. Taken together, the data suggest that premexetred and sorafenib act synergistically to enhance tumor killing via the promotion of a toxic form of autophagy that leads to activation of the intrinsic apoptosis pathway, and predict that combination treatment represents a future therapeutic option in the treatment of solid tumors.
doi:10.1158/0008-5472.CAN-11-0898
PMCID: PMC3139015  PMID: 21622715
4.  Regulation of autophagy by ceramide-CD95-PERK signaling 
Autophagy  2008;4(7):929-931.
The manuscripts by Park et al.1 and Zhang et al.2 were initially planned as studies to understand the regulation of cell survival in transformed cells treated with sorafenib and vorinostat, and in primary hepatocytes treated with a bile acid+MEK1/2 inhibitor. In both cell systems we discovered that the toxicity of sorafenib and vorinostat or bile acid+MEK1/2 inhibitor exposure depended on the generation of ceramide and the ligand-independent activation of the CD95 death receptor, with subsequent activation of pro-caspase 8. We noted, however, in these systems that, in parallel with death receptor–induced activation of the extrinsic pathway, CD95 signaling also promoted increased phosphorylation of PKR-like endoplasmic reticulum kinase (PERK) and eIF2α, increased expression of ATG5, and increased processing of LC3 and vesicularization of a GFP-LC3 construct. The knockdown of ATG5 expression blocked GFP-LC3 vesicularization and enhanced cell killing. Thus ceramide-CD95 signaling promoted cell death via activation of pro-caspase 8 and cell survival via autophagy. PERK was shown to signal in a switch-hitting fashion; PERK promoted CD95-DISC formation and an eIF2α-dependent reduction in c-FLIP-s levels that were essential for cell killing to proceed, but in parallel it also promoted autophagy that was protective. The death receptor-induced apoptosis and autophagy occur proximal to the receptor rather than the mitochondrion, and the relative flow of death receptor signaling into either pathway may determine cell fate. Finally, death receptor induced apoptosis and autophagy could be potential targets for therapeutic intervention.
PMCID: PMC3292039  PMID: 18719356
Vorinostat; Sorafenib; bile acid; CD95; autophagy; ceramide; cell death; ASMase
5.  OSU-03012 enhances Ad.mda-7-induced GBM cell killing via ER stress and autophagy and by decreasing expression of mitochondrial protective proteins 
Cancer biology & therapy  2010;9(7):526-536.
The present studies focused on determining whether the autophagy-inducing drug OSU-03012 (AR-12) could enhance the toxicity of recombinant adenoviral delivery of melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) in glioblastoma multiforme (GBM) cells. The toxicity of a recombinant adenovirus to express MDA-7/IL-24 (Ad.mda-7) was enhanced by OSU-03012 in a diverse panel of primary human GBM cells. The enhanced toxicity correlated with reduced ERK1/2 phosphorylation and expression of MCL-1 and BCL-XL, and was blocked by molecular activation of ERK1/2 and by inhibition of the intrinsic, but not the extrinsic, apoptosis pathway. Both OSU-03012 and expression of MDA-7/IL-24 increased phosphorylation of PKR-like endoplasmic reticulum kinase (PERK) that correlated with increased levels of autophagy and expression of dominant negative PERK blocked autophagy induction and tumor cell death. Knockdown of ATG5 or Beclin1 suppressed OSU-03012 enhanced MDA-7/IL-24-induced autophagy and blocked the lethal interaction between the two agents. Ad.mda-7-infected GBM cells secreted MDA-7/IL-24 into the growth media and this conditioned media induced expression of MDA-7/IL-24 in uninfected GBM cells. OSU-03012 interacted with conditioned media to kill GBM cells and knockdown of MDA-7/IL-24 in these cells suppressed tumor cell killing. Collectively, our data demonstrate that the induction of autophagy and mitochondrial dysfunction by a combinatorial treatment approach represents a potentially viable strategy to kill primary human GBM cells.
PMCID: PMC2888700  PMID: 20107314
ROS; caspase; ER stress; CD95; cell death
6.  The development of MDA-7/IL-24 as a cancer therapeutic 
Pharmacology & therapeutics  2010;128(2):375-384.
The cytokine melanoma differentiation associated gene 7 (mda-7) was identified by subtractive hybridization as a protein whose expression increased during the induction of terminal differentiation, and that was either not expressed or was present at low levels in tumor cells compared to non-transformed cells. Based on conserved structure, chromosomal location and cytokine-like properties, MDA-7, was classified as a member of the interleukin (IL)-10 gene family and designated as MDA-7/IL-24. Multiple studies have demonstrated that expression of MDA-7/IL-24 in a wide variety of tumor cell types, but not in corresponding equivalent non-transformed cells, causes their growth arrest and rapid cell death. In addition, MDA-7/IL-24 has been noted to radiosensitize tumor cells which in part is due to the generation of reactive oxygen species (ROS) and ceramide that cause endoplasmic reticulum stress and suppress protein translation. Phase I clinical trial data has shown that a recombinant adenovirus expressing MDA-7/IL-24 (Ad.mda-7 (INGN-241)) was safe and had measurable tumoricidal effects in over 40% of patients, strongly arguing that MDA-7/IL-24 could have significant therapeutic value. This review describes what is presently known about the impact of MDA-7/IL-24 on tumor cell biology and its potential therapeutic applications.
doi:10.1016/j.pharmthera.2010.08.001
PMCID: PMC2947573  PMID: 20732354
MDA-7; IL-24; Apoptosis; Autophagy; Ceramide; ROS; Ca2+; Clinical trial; Signal transduction; PERK; ER stress; MCL-1
7.  Caspase-, cathepsin-, and PERK-dependent regulation of MDA-7/IL-24-induced cell killing in primary human glioma cells 
Molecular cancer therapeutics  2008;7(2):297-313.
Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is a novel cytokine displaying selective apoptosis-inducing activity in transformed cells without harming normal cells. The present studies focused on defining the mechanism(s) by which a GST-MDA-7 fusion protein inhibits cell survival of primary human glioma cells in vitro. GST-MDA-7 killed glioma cells with diverse genetic characteristics that correlated with inactivation of ERK1/2 and activation of JNK1-3. Activation of JNK1-3 was dependent on protein kinase R–like endoplasmic reticulum kinase (PERK), and GST-MDA-7 lethality was suppressed in PERK−/− cells. JNK1-3 signaling activated BAX, whereas inhibition of JNK1-3, deletion of BAX, or expression of dominant-negative caspase-9 suppressed lethality. GST-MDA-7 also promoted a PERK-, JNK-, and cathepsin B–dependent cleavage of BID; loss of BID function promoted survival. GST-MDA-7 suppressed BAD and BIM phosphorylation and heat shock protein 70 (HSP70) expression. GST-MDA-7 caused PERK-dependent vacuolization of LC3-expressing endosomes whose formation was suppressed by incubation with 3-methylade-nine, expression of HSP70 or BiP/GRP78, or knockdown of ATG5 or Beclin-1 expression but not by inhibition of the JNK1-3 pathway. Knockdown of ATG5 or Beclin-1 expression or overexpression of HSP70 reduced GST-MDA-7 lethality. Our data show that GST-MDA-7 induces an endoplasmic reticulum stress response that is causal in the activation of multiple proapoptotic pathways, which converge on the mitochondrion and highlight the complexity of signaling pathways altered by mda-7/IL-24 in glioma cells that ultimately culminate in decreased tumor cell survival.
doi:10.1158/1535-7163.MCT-07-2166
PMCID: PMC3204355  PMID: 18281515
8.  mda-7/IL-24: A Unique Member of the IL-10 Gene Family Promoting Cancer-Targeted Toxicity 
Cytokine & growth factor reviews  2010;21(5):381-391.
Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) is a unique member of the IL-10 gene family that displays nearly ubiquitous cancer-specific toxicity, with no harmful effects toward normal cells or tissues. mda-7/IL-24 was cloned from human melanoma cells by differentiation induction subtraction hybridization (DISH) and promotes endoplasmic reticulum (ER) stress culminating in apoptosis or toxic autophagy in a broad-spectrum of human cancers, when assayed in cell culture, in vivo in human tumor xenograft mouse models and in a Phase I clinical trial in patients with advanced cancers. This therapeutically active cytokine also induces indirect anti-tumor activity through inhibition of angiogenesis, stimulation of an anti-tumor immune response, and sensitization of cancer cells to radiation-, chemotherapy- and antibody-induced killing.
doi:10.1016/j.cytogfr.2010.08.004
PMCID: PMC3164830  PMID: 20926331
mda-7/IL-24; apoptosis; autophagy; bystander antitumor activity; cancer terminator virus
9.  MDA-7/IL-24 as a cancer therapeutic: from bench to bedside 
Anti-cancer drugs  2010;21(8):725-731.
The novel cytokine melanoma differentiation associated gene-7 (mda-7) was identified by subtractive hybridization in the mid-1990s as a protein whose expression increased during the induction of terminal differentiation, and that was either not expressed or was present at low levels in tumor cells compared to non-transformed cells. Based on conserved structure, chromosomal location and cytokine-like properties, MDA-7, has now been classified as a member of the expanding interleukin (IL)-10 gene family and designated as MDA-7/IL-24. Multiple studies have demonstrated that expression of MDA-7/IL-24 in a wide variety of tumor cell types, but not in corresponding equivalent non-transformed cells, causes their growth arrest and ultimately cell death. In addition, MDA-7/IL-24 has been noted to be a radiosensitizing cytokine, which in part is due to the generation of reactive oxygen species (ROS) and ceramide that cause endoplasmic reticulum stress. Phase I clinical trial data has shown that a recombinant adenovirus expressing MDA-7/IL-24 (Ad.mda-7 (INGN-241)) was safe and had measurable tumoricidal effects in over 40% of patients, which strongly argues that MDA-7/IL-24 may have significant therapeutic value. This review describes what is known about the impact of MDA-7/IL-24 on tumor cell biology and its potential therapeutic applications.
doi:10.1097/CAD.0b013e32833cfbe1
PMCID: PMC2915543  PMID: 20613485
MDA-7: melanoma differentiation associated gene 7
10.  Vorinostat and sorafenib increase CD95 activation in gastrointestinal tumor cells through a Ca2+ - de novo ceramide - PP2A - ROS dependent signaling pathway 
Cancer research  2010;70(15):6313-6324.
The targeted therapeutics sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95, and this drug combination is entering phase I evaluation. In this study we determined how CD95 is activated by treatment with this drug combination. Low doses of sorafenib and vorinostat but not the individual drugs rapidly increased ROS, Ca2+ and ceramide levels in GI tumor cells. The production of ROS was reduced in Rho zero cells. Quenching ROS blocked drug-induced CD95 surface localization and apoptosis. ROS generation, CD95 activation and cell killing was also blocked by quenching of induced Ca2+ levels or by inhibition of PP2A. Inhibition of acidic sphingomyelinase or de novo ceramide generation blocked the induction of ROS however combined inhibition of both acidic sphingomyelinase and de novo ceramide generation was required to block the induction of Ca2+. Quenching of ROS did not impact on drug-induced ceramide/dihydro-ceramide levels whereas quenching of Ca2+ reduced the ceramide increase. Sorafenib and vorinostat treatment radiosensitized liver and pancreatic cancer cells, an effect that was suppressed by quenching ROS or knock down of LASS6. Further, sorafenib and vorinostat treatment suppressed the growth of pancreatic tumors in vivo. Our findings demonstrate that induction of cytosolic Ca2+ by sorafenib and vorinostat is a primary event that elevates dihydroceramide levels, each essential steps in ROS generation that promotes CD95 activation.
doi:10.1158/0008-5472.CAN-10-0999
PMCID: PMC2918282  PMID: 20631069
11.  Sorafenib activates CD95 and promotes autophagy and cell death via Src family kinases in GI tumor cells 
Molecular cancer therapeutics  2010;9(8):2220-2231.
Sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95, and the present studies have determined individually how sorafenib and vorinostat contribute to CD95 activation. Sorafenib (3-6 μM) promoted a dose-dependent increase in Src Y416, ERBB1 Y845 and CD95 Y232/Y291 phosphorylation, and Src Y527 dephosphorylation. Low levels of sorafenib (3 μM) –induced CD95 tyrosine phosphorylation did not promote surface localization whereas sorafenib (6 μM), or sorafenib (3 μM) and vorinostat (500 nM) treatment promoted higher levels of CD95 phosphorylation that correlated with DISC formation, receptor surface localization and autophagy. CD95 (Y232F, Y291F) was not tyrosine phosphorylated and was unable to plasma membrane localize or induce autophagy. Knock down / knock out of Src family kinases abolished sorafenib –induced: CD95 tyrosine phosphorylation; DISC formation; and the induction of cell death and autophagy. Knock down of PDGFRβ enhanced Src Y416 and CD95 tyrosine phosphorylation that correlated with elevated CD95 plasma membrane levels and autophagy, and with a reduced ability of sorafenib to promote CD95 membrane localization. Vorinostat increased ROS levels; and in a delayed NFκB-dependent fashion, those of FAS ligand and CD95. Neutralization of FAS-L did not alter the initial rapid drug-induced activation of CD95 however, neutralization of FAS-L reduced sorafenib + vorinostat toxicity by ~50%. Thus sorafenib contributes to CD95 activation by promoting receptor tyrosine phosphorylation whereas vorinostat contributes to CD95 activation via initial facilitation of ROS generation and subsequently of FAS-L expression.
doi:10.1158/1535-7163.MCT-10-0274
PMCID: PMC2933415  PMID: 20682655
Vorinostat; Sorafenib; CD95; c-FLIP-s; FAS-L; cell death; autophagy
12.  17AAG and MEK1/2 inhibitors kill GI tumor cells via Ca2+-dependent suppression of GRP78/BiP and induction of ceramide and ROS 
Molecular cancer therapeutics  2010;9(5):1378-1395.
The present studies determined in greater detail the molecular mechanisms upstream of the CD95 death receptor by which geldanamycin HSP90 inhibitors and MEK1/2 inhibitors interact to kill carcinoma cells. MEK1/2 inhibition enhanced 17AAG toxicity that was suppressed in cells deleted for mutant active RAS which were non-tumorigenic but was magnified in isogenic tumorigenic cells expressing H-RAS V12 or K-RAS D13. MEK1/2 inhibitor and 17AAG treatment increased intracellular Ca2+ levels and reduced GRP78/BiP expression in a Ca2+ -dependent manner. GRP78/BiP over-expression, however, also suppressed drug-induced intracellular Ca2+ levels. MEK1/2 inhibitor and 17AAG treatment increased ROS levels that were blocked by quenching Ca2+ or over-expression of GRP78/BiP. MEK1/2 inhibitor and 17AAG treatment activated CD95 and inhibition of ceramide synthesis; ROS or Ca2+ quenching blocked CD95 activation. In SW620 cells that are patient matched to SW480 cells, MEK1/2 inhibitor and 17AAG toxicity was significantly reduced that correlated with a lack of CD95 activation and lower expression of ceramide synthase 6 (LASS6). Over-expression of LASS6 in SW620 cells enhanced drug-induced CD95 activation and enhanced tumor cell killing. Inhibition of ceramide signaling abolished drug-induced ROS generation but not drug-induced cytosolic Ca2+ levels. Thus treatment of tumor cells with MEK1/2 inhibitor and 17AAG induces cytosolic Ca2+ and loss of GRP78/BiP function, leading to de novo ceramide synthesis pathway activation that plays a key role in ROS generation and CD95 activation.
doi:10.1158/1535-7163.MCT-09-1131
PMCID: PMC2868106  PMID: 20442308
Geldanamycin; 17AAG; MEK1/2 inhibitor; CD95; c-FLIP-s; GRP78/BiP; autophagy; cell death; ASMase; de novo
13.  Enhancing mda-7/IL-24 therapy in renal carcinoma cells by inhibiting multiple protective signaling pathways using sorafenib and by Ad.5/3 gene delivery 
Cancer Biology & Therapy  2010;10(12):1290-1305.
We have determined whether an adenovirus that comprises the tail and shaft domains of a serotype 5 virus and the knob domain of a serotype 3 virus expressing MDA-7/IL-24, Ad.5/3-mda-7, more effectively infects and kills renal carcinoma cells (RCCs) compared to a serotype 5 virus, Ad.5-mda-7. RCCs are a tumor cell type that generally does not express the receptor for the type 5 adenovirus; the coxsakie and adenovirus receptor (CAR). Ad.5/3-mda-7 infected RCCs to a much greater degree than Ad.5-mda-7. MDA-7/IL-24 protein secreted from Ad.5/3-mda-7-infected RCCs induced MDA-7/IL-24 expression and promoted apoptosis in uninfected “bystander” RCCs. MDA-7/IL-24 killed both infected and bystander RCCs via CD95 activation. Knockdown of intracellular MDA-7/IL-24 in uninfected RCCs blocked the lethal effects of conditioned media. Infection of RCC tumors in one flank, with Ad.5/3-mda-7, suppressed growth of infected tumors and reduced the growth rate of uninfected tumors implanted on the opposite flank. The toxicity of the serotype 5/3 recombinant adenovirus to express MDA-7/IL-24 was enhanced by combined molecular or small molecule inhibition of MEK1/2 and PI3K; inhibition of mTOR, PI3K and MEK1/2; or use of the multi-kinase inhibitor sorafenib. In RCCs, combined inhibition of cytoprotective cell signaling pathways enhanced the MDA-7/IL-24-induction of CD95 activation, with greater mitochondrial dysfunction due to loss of MCL-1 and BCL-XL expression and tumor cell death. Treatment of RCC tumors in vivo with sorafenib also enhanced Ad.5/3-mda-7 toxicity and prolonged animal survival. Future combinations of these approaches hold promise for developing a more effective therapy for kidney cancer.
doi:10.4161/cbt.10.12.13497
PMCID: PMC3047088  PMID: 20948318
ERK; JNK; PI3K; AKT; MDA-7/IL-24; sorafenib; PERK; MAPK; interleukin; RCC; kidney
14.  MDA-7/IL-24–induced cell killing in malignant renal carcinoma cells occurs by a ceramide/CD95/PERK–dependent mechanism 
Molecular cancer therapeutics  2009;8(5):1280-1291.
Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) is a novel cytokine displaying selective apoptosis-inducing activity in transformed cells without harming normal cells. The present studies focused on clarifying the mechanism(s) by which glutathione S-transferase (GST)-MDA-7 altered cell survival of human renal carcinoma cells in vitro. GST-MDA-7 caused plasma membrane clustering of CD95 and the association of CD95 with procaspase-8. GST-MDA-7 lethality was suppressed by inhibition of caspase-8 or by overexpression of short-form cellular FLICE inhibitory protein, but only weakly by inhibition of cathepsin proteases. GST-MDA-7–induced CD95 clustering (and apoptosis) was blocked by knockdown of acidic sphingomyelinase or, to a greater extent, ceramide synthase-6 expression. GST-MDA-7 killing was, in parallel, dependent on inactivation of extracellular signal–regulated kinase 1/2 and on CD95-induced p38 mitogen-activated protein kinase and c-jun NH2-terminal kinase-1/2 signaling. Knockdown of CD95 expression abolished GST-MDA-7–induced phosphorylation of protein kinase R–like endoplasmic reticulum kinase. GST-MDA-7 lethality was suppressed by knockout or expression of a dominant negative protein kinase R–like endoplasmic reticulum kinase that correlated with reduced c-jun NH2-terminal kinase-1/2 and p38 mitogen-activated protein kinase signaling and maintained extracellular signal–regulated kinase-1/2 phosphorylation. GST-MDA-7 caused vacuolization of LC3 through a mechanism that was largely CD95 dependent and whose formation was suppressed by knockdown of ATG5 expression. Knockdown of ATG5 suppressed GST-MDA-7 toxicity. Our data show that in kidney cancer cells GST-MDA-7 induces ceramide-dependent activation of CD95, which is causal in promoting an endoplasmic reticulum stress response that activates multiple proapoptotic pathways to decrease survival.
doi:10.1158/1535-7163.MCT-09-0073
PMCID: PMC2889018  PMID: 19417161
15.  Vorinostat and sorafenib increase ER stress, autophagy and apoptosis via ceramide-dependent CD95 and PERK activation 
Cancer biology & therapy  2008;7(10):1648-1662.
We recently noted that low doses of sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95, and this drug combination is entering phase I trials. The present studies mechanistically extended our initial observations. Low doses of sorafenib and vorinostat, but not the individual agents, caused an acidic sphingomyelinase and fumonisin B1-dependent increase in CD95 surface levels and CD95 association with caspase 8. Knock down of CD95 or FADD expression reduced sorafenib/vorinostat lethality. Signaling by CD95 caused PERK activation that was responsible for both promoting caspase 8 association with CD95 and for increased eIF2α phosphorylation; suppression of eIF2α function abolished drug combination lethality. Cell killing was paralleled by PERK- and eIF2α-dependent lowering of c-FLIP-s protein levels and over-expression of c-FLIP-s maintained cell viability. In a CD95-, FADD- and PERK-dependent fashion, sorafenib and vorinostat increased expression of ATG5 that was responsible for enhanced autophagy. Expression of PDGFRβ and FLT3 were essential for high dose single agent sorafenib treatment to promote autophagy. Suppression of PERK function reduced sorafenib and vorinostat lethality whereas suppression of ATG5 levels elevated sorafenib and vorinostat lethality. Over-expression of c-FLIP-s blocked apoptosis and enhanced drug-induced autophagy. Thus sorafenib and vorinostat promote ceramide-dependent CD95 activation followed by induction of multiple downstream survival regulatory signals: ceramide-CD95-PERK-FADD-pro-caspase 8 (death); ceramide-CD95-PERK-eIF2α -↓c-FLIP-s (death); ceramide-CD95-PERK-ATG5-autophagy (survival).
PMCID: PMC2674577  PMID: 18787411
Vorinostat; Sorafenib; CD95; c-FLIP-s; PDGFRβ; FLT3; autophagy; ceramide; cell death; ASMase
16.  MEK1/2 inhibitors and 17AAG synergize to kill human GI tumor cells in vitro via suppression of c-FLIP-s levels and activation of CD95 
Molecular cancer therapeutics  2008;7(9):2633-2648.
Prior studies have noted that inhibitors of MEK1/2 enhanced geldanamycin lethality in malignant hematopoietic cells by promoting mitochondrial dysfunction. The present studies focused on defining the mechanism(s) by which these agents altered survival in carcinoma cells. MEK1/2 inhibitors (PD184352; AZD6244 (ARRY-142886)) interacted in a synergistic manner with geldanamycins (17AAG, 17DMAG) to kill hepatoma and pancreatic carcinoma cells that correlated with inactivation of ERK1/2 and AKT and with activation of p38 MAPK; p38 MAPK activation was ROS-dependent. Treatment of cells with MEK1/2 inhibitors and 17AAG reduced expression of c-FLIP-s that was mechanistically connected to loss of MEK1/2 and AKT function; inhibition of caspase 8 or over-expression of c-FLIP-s abolished cell killing by MEK1/2 inhibitors and 17AAG. Treatment of cells with MEK1/2 inhibitors and 17AAG caused a p38 MAPK-dependent plasma membrane clustering of CD95 without altering the levels or cleavage of FAS ligand. In parallel, treatment of cells with MEK1/2 inhibitors and 17AAG caused a p38 MAPK-dependent association of caspase 8 with CD95. Inhibition of p38 MAPK or knock down of BID, FADD or CD95 expression suppressed MEK1/2 inhibitor and 17AAG lethality. Similar correlative data were obtained using a xenograft flank tumor model system. Our data demonstrate that treatment of tumor cells with MEK1/2 inhibitors and 17AAG induces activation of the extrinsic pathway and that suppression of c-FLIP-s expression is crucial in transduction of the apoptotic signal from CD95 to promote cell death.
doi:10.1158/1535-7163.MCT-08-0400
PMCID: PMC2585522  PMID: 18790746
CD95; caspase; extrinsic; FLIP
17.  Lapatinib resistance in HCT116 cells is mediated by elevated MCL-1 expression, decreased BAK activation, and not by ERBB receptor mutation 
Molecular pharmacology  2008;74(3):807-822.
We have defined some of the mechanisms by which the kinase inhibitor Lapatinib kills HCT116 cells. Lapatinib inhibited radiation-induced activation of ERBB1/2, ERK1/2 and AKT, and radiosensitized HCT116 cells. Prolonged incubation of HCT116 cells with Lapatinib caused cell killing followed by outgrowth of Lapatinib adapted cells. Adapted cells were resistant to serum-starvation –induced cell killing and were cross resistant to multiple therapeutic drugs. Lapatinib was competent to inhibit basal and EGF-stimulated ERBB1 phosphorylation in adapted cells. Co-expression of dominant negative ERBB1 and dominant negative ERBB2 inhibited basal and EGF-stimulated ERBB1 and ERBB2 phosphorylation in parental cells. However in neither parental nor adapted cells did expression of dominant negative ERBB1 and dominant negative ERBB2 recapitulate the cell death promoting effects of Lapatinib. Adapted cells had increased expression of MCL-1, decreased expression of BAX, and decreased activation of BAX and BAK. Over-expression of BCL-XL protected parental cells from Lapatinib toxicity. Knock down of MCL-1 expression enhanced Lapatinib toxicity in adapted cells that was reverted by knock down of BAK expression. Inhibition of caspase function modestly reduced Lapatinib toxicity in parental cells whereas knock down of AIF expression suppressed Lapatinib toxicity. Thus in HCT116 cells Lapatinib adaptation can be mediated by altered expression of pro- and anti-apoptotic proteins that maintain mitochondrial function.
doi:10.1124/mol.108.047365
PMCID: PMC2574656  PMID: 18544666
Lapatinib; Ras; cell death
18.  Vorinostat and sorafenib synergistically kill tumor cells via FLIP suppression and CD95 activation 
Purpose and Design
Mechanism(s) by which the multi-kinase inhibitor sorafenib and the histone deacetylase inhibitor vorinostat interact to kill hepatic, renal and pancreatic adenocarcinoma cells have been defined.
Results
Low doses of sorafenib and vorinostat interacted in vitro in a synergistic fashion to kill hepatic, renal and pancreatic adenocarcinoma cells in multiple short term viability (24–96h) and in long term colony formation assays. Cell killing was suppressed by inhibition of cathepsin proteases and caspase 8, and to a lesser extent by inhibition of caspase 9. Twenty four hours after exposure, the activities of ERK1/2, AKT and NFκB were only modestly modulated by sorafenib and vorinostat treatment. However, 24h after exposure, sorafenib and vorinostat- treated cells exhibited markedly diminished expression of c-FLIP-s, full length BID, BCL-2, BCLXL, MCL-1, XIAP, increased expression of BIM, and increased activation of BAX, BAK and BAD. Expression of eIF2α S51A blocked sorafenib and vorinostat –induced suppression of c-FLIP-s levels and over-expression of c-FLIP-s abolished lethality. Sorafenib and vorinostat treatment increased surface levels of CD95 and CD95 association with caspase 8. Knock down of CD95 or FADD expression significantly reduced sorafenib / vorinostat -mediated lethality.
Conclusions
These data demonstrate that combined exposure of epithelial tumor cell types to sorafenib and vorinostat diminishes expression of multiple anti-apoptotic proteins, promotes activation of the CD95 extrinsic apoptotic and the lysosomal protease pathways, and that suppression of c-FLIP-s expression represents a critical event in transduction of the pro-apoptotic signals from CD95 to promote mitochondrial dysfunction and death.
doi:10.1158/1078-0432.CCR-08-0469
PMCID: PMC2561272  PMID: 18765530
Vorinostat; Sorafenib; CD95; c-FLIP-s; caspase 8; cathepsin; cell death
19.  PERK-dependent regulation of MDA-7/IL-24-induced autophagy in primary human glioma cells 
Autophagy  2008;4(4):513-515.
Melanoma differentiation associated gene-7/interleukin 24 (mda-7/IL-24) is a novel cytokine displaying selective apoptosis-inducing activity in transformed cells without harming normal cells. The studies by further defines the mechanism(s) by which a GST-MDA-7 fusion protein inhibits cell survival of primary human glioma cells in vitro. GST-MDA-7 killed glioma cells with diverse genetic characteristics that were dependent on activation of JNK1-3 with subsequent activation of BAX and the induction of mitochondrial dysfunction. Activation of JNK1-3 was dependent upon protein kinase R-like endoplasmic reticulum kinase (PERK) and GST-MDA-7 lethality was suppressed in PERK-/- cells. GST-MDA-7 caused PERK-dependent vacuolization of LC3-expressing endosomes whose formation was suppressed by incubation with 3-methyladenine, expression of HSP70 or of BiP/GRP78, or by knockdown of ATG5 or Beclin 1 expression, but not by inhibition of the JNK1-3 pathway. Knockdown of ATG5 or Beclin 1 expression or overexpression of HSP70 reduced GST-MDA-7 lethality. Our data demonstrate that GST-MDA-7 induces an ER stress response that, via the induction of autophagy, is causal in the activation of pro-apoptotic pathways that converge on the mitochondrion and ultimately culminate in decreased glioma cell survival.
PMCID: PMC2674579  PMID: 18299661
autophagy; caspase; ER stress; cell death
20.  OSU-03012 Stimulates PKR-Like Endoplasmic Reticulum-Dependent Increases in 70-kDa Heat Shock Protein Expression, Attenuating Its Lethal Actions in Transformed Cells 
Molecular pharmacology  2008;73(4):1168-1184.
We have further defined mechanism(s) by which 2-amino-N-{4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-phenyl}acetamide [OSU-03012 (OSU)], a derivative of the cyclooxygenase-2 (COX2) inhibitor celecoxib but lacking COX2 inhibitory activity, kills transformed cells. In cells lacking expression of protein kinase R-like endoplasmic reticulum kinase (PERK-/-), the lethality of OSU was attenuated. OSU enhanced the expression of Beclin 1 and ATG5 and cleavage of pro-caspase 4 in a PERK-dependent fashion and promoted the Beclin 1- and ATG5-dependent formation of vacuoles containing LC3, followed by a subsequent caspase 4-dependent cleavage of cathepsin B and a cathepsin B-dependent formation of low pH intracellular vesicles; cathepsin B was activated and released into the cytosol and genetic suppression of caspase 4, cathepsin B, or apoptosis-inducing factor function significantly suppressed cell killing. In parallel, OSU caused PERK-dependent increases in 70-kDa heat shock protein (HSP70) expression and decreases in 90-kDa heat shock protein (HSP90) and Grp78/BiP expression. Changes in HSP70 expression were post-transcriptional. Knockdown or small-molecule inhibition of HSP70 expression enhanced OSU toxicity, and overexpression of HSP70 suppressed OSU-induced low pH vesicle formation and lethality. Our data demonstrate that OSU-03012 causes cell killing that is dependent on PERK-induced activation of multiple toxic proteases. OSU-03012 also increased expression of HSP70 in a PERK-dependent fashion, providing support for the contention that OSU-03012-induced PERK signaling promotes both cell survival and cell death processes.
doi:10.1124/mol.107.042697
PMCID: PMC2674576  PMID: 18182481

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